Process for the determination of catecholamine and serotonin metabolites

ABSTRACT

Chromatograms of high definition and good separation for the determination of catecholamine and serotonin metabolites in body fluids, e.g., urine, are obtained using as the adsorbent chromatographic grade cellulose impregnated with polyethylenimine. The chromatogram is preferably developed using a multicomponent solvent mixture containing water, a solvent substantially immiscible in water, a solvent miscible with water and the substantially water-immiscible solvent, and an acid, e.g., chloroform/n-butanol/ethanol/glacial acetic acid/water in a 5/55/10/15/15 volume ratio.

llnited States Patent raffczyk et a]. [451 Apr. 18, 1972 [54] PROCESS FOR THE DETERMINATION [56] References Cited OF CATECHOLAMINE AND SEROTONIN METABOLITES UNTEDSTjTESPATENTS Inventors: Friedrich y Roland g both 3,562,289 2/ 1971 Battista et al. ..210/ 198 of Darmstadt Germany Primary Examiner-J. L. De Cesare [73] Assignee: Merck Patent Gesellschaft mit AlmmeyMllle", Raptes & whlte beschrankter Haftung, Darmstadt, Germany [57] ABSTRACT [22] Fil d; S 9, 1970 Chromatograms of high definition and good separation for the determination of catecholamine and serotonin metabolites in PP 70,929 body fluids, e.g., urine, are obtained using as the adsorbent chromatographic grade cellulose impregnated with [30] Foreign Application p i Data polyethylenimine. The chromatogram is preferably developed using a multicomponent solvent mixture containing water, a p 1969 Germany 19 45 471-8 solvent substantially immiscible in water, a solvent miscible with water and the substantially water-immiscible solvent, and UeS- an acid e g chloroform/n.butanol/ethanol/glacia] acetic [51] Int. Cl ..B01d 15/08 acid/water in 3 5/55 10 5/15 volume m; [58] Field of Search ..210/3l, 198

19 Claims, No Drawings PROCESS FOR THE DETERMINATION OF CATECIIOLAMINE AND SEROTONIN METABOLITES determination of catecholarnine and serotonin metabolites in the urine.

Catecholamines are understood in clinical chemistry to mean noradrenaline (NA), adrenaline (A), and dopamine. The metabolites thereof which are of greatest importance with respect to quantity and, at present, with respect to diagnosis of diseases, are metadrenaline, norrnetadrenaline and the phenolcarboxylic acids 4-hydroxy-3-methoxymandelic acid (HMMS or VMS vanillin mandelic acid), vanillic acid (VS) and homovanillic acid (I-IVS). The excretion of these metabolites is increased in various diseases, for example in case of pheochromocytoma, neuroblastoma and ganglioneuroma.

The serotonin metabolite most important from a diagnostic view point is 5-hydroxyindoleacetic acid (HIES). For example, the excretion of I-IIES is greatly increased in cases of bronchus carcinoma and small intestine carcinoid.

Numerous methods exist for detecting these metabolites. The fluorometric and spectrophotometric methods, presently employed most frequently, exhibit certain disadvantages. They are either relatively simple but unspecific and thus of minor value in diagnoses, or specific but their use is very complicated and involves a large amount of work.

In a conventional process which uses cation exchangers and subsequent alkaline treatment, the metabolites bearing an amino group, metadrenaline (metanephrine), as well as normetadrenaline (normetanephrine), can be detected. However, these metabolites are excreted to an increased degree only in case of an attack.

' In contrast thereto, HMMS is excreted to an increased extent also during the attack-free interval. However, the simultaneous detection of IIMMS is difficult, because numerous other phenols are also present in the urine, which phenols form colors with the respective reagents, and because the basic ion exchangers do not bind the phenolcarboxylic acids to a particularly great extent in the presence of the uric salts.

Purely chromatographic methods exhibit the advantage over processes operating with the aid of cation exchangers in that several metabolites can be simultaneously and specifically determined side-by-side. From the literature, several methods using paper and thin-layer chromatography are known for the determination of these metabolites. In these conventional processes, the extraction of the urine is generally followed by the steps of drying, filtering, and concentrating the thus-obtained extract. Thereafter, a separation by paper or thin-layer chromatography is conducted on silica gel or cellulose. The detection of the metabolites is effected in all cases in a conventional manner with a color reagent, the color being compared with a standard.

It has been suggested to omit the pretreatment of the urine prior to applying same to a cellulose layer. Although this represents an essential simplification of the overall diagnostic process, only unsatisfactory separations are possible, since other urine components, e.g., salts and urea, distort the separated zones. Also, only HMMS can be detected and its identification is relatively uncertain. Quantitative I-IMMS estimation contains a large factor of uncertainty and only highly excessive excretions of I-IMMS can be determined. The lower limit of detection of I-IMMS is about 10 mg. percent per day. Since the normal level of excretion is 3-4 mg. percent, values about or below 10 mg. percent per day should also be detected.

It has now been discovered that by the use of a particular chromatographic adsorbent, surprisingly it is possible to obtain flawless chromatograms with good zone resolution sharpness and separation. Very surprisingly, the urine can be appliedwithout any pretreatment directly to the adsorbent. Furthermore, the lower limits of detection of the metabolites are very low. The process of this invention provides an especially advantageous process for the simultaneous determination of the catecholamine and serotonin metabolites. The novel process is extremely simple to conduct and includes in its range of detection all metabolites which are of diagnostic importance. Moreover, the identification and quantitative detemrination of the metabolites separated by chromatography can be effected with great certainty.

SUMMARY OF THE INVENTION According to this invention, catecholamine and serotonin metabolites in body fluids are determined by separation by conventional thin-layer chromatography and color development of the chromatogram employing as chromatographic adsorbent cellulose impregnated with polyethylenimine. The chromatogram is developed preferably using a solvent mixture described hereinafter.

DETAILED DISCUSSION The chromatographic adsorbent employed preferably is conventional cellulose impregnated with polyethylenimine for chromatographic purposes (PEI cellulose). It is also possible to impregnate any cellulose of a quality suitable for chromatographic purposes, preferably micro-crystalline cellulose, with polyethylenimine in a conventional manner. See, for example, Biochimica Biophysica Acta, Vol.61, pp.852-854 (1962). This is most simply done using a commercially available polyethylenimine solution which is about 50 percent aqueous. Such a solution is thoroughly mixed with the cellulose, optionally after dilution with deionized water. The thus-obtained suspension can be used as such for coating the thin-layer chromatographic plates in the conventional manner. The proportion of polyethylenimine to adsorbent is usually about 0.15 to 10 percent by weight, preferably about 0.5 to 3.5 percent.

The polyethylenimine-impregnated cellulose should not be a PEI cellulose neutralized with hydrochloric acid, which is the form customarily employed in chromatography. It is essential that a PEI cellulose is employed which does not contain a firmly bound counter-ion. For this reason, the polyethylenimine should be adsorbed by the cellulose in free form, i.e., as the free base, or if it is adsorbed in neutralized form, it should contain only those anions which have a selectivity coefficient lower than the chloride anion, e.g., forrnate, acetate, propionate and butyrate ions. As is known, selectivity coefficient is a measure of the relative affinity of the ions with respect to the ion exchanger.

The composition of the eluent employed to develop the chromatogram affects the separation achieved on the chromatograms. The eluent mixture preferably consists of at least three components. Suitably, the eluent, i.e., mobile phase, contains, in addition to at least 5 percent of water, at least one other solvent from each of the following groups:

A. solvents immiscible or miscible to only an insubstantial extent with water;

B. solvents miscible both with water and with organic solvents;

C. acids, preferably lower alkanecarboxylic acids.

It is especially important, at least for the first chromatographic separation, to employ an acidic multicomponent mixture, preferably consisting of at least three components. The addition of at least 5 percent by volume of water, preferably between 5 and 20 percent, is especially advantageous. The desired separations can be obtained especially advantageously if eluents are employed containing, in addition to water, at least one solvent which is immiscible or miscible to only an insubstantial extent with water; at least one solvent miscible with both water and with organic solvents; and at least one acid, preferably a lower-alkanoic acid.

Examples of solvents which are immiscible with water, or miscible only to an insubstantial extent (group A) are chlorinated hydrocarbons, e.g., chloroform and methylene chloride; aliphatic alcohols containing more than three carbon atoms, e.g., butanols (except tert.butyl alcohol) and amyl alcohols; esters, e.g., esters of acetic acid and other loweraliphatic carboxylic acids, preferably esters of acetic acid, e.g., butyl acetate, and isoamyl acetate; ethers, e.g., diethyl ether; and aromatic hydrocarbons, e.g., benzene, toluene and xylene.

Examples of suitable solvents which are miscible both with water and organic solvents (group B) are lower-aliphatic alcohols, particularly methanol, ethanol, nand iso-propanol; ketones, e.g., dialkyl ketones, especially acetone and methyl ethyl ketone, cycloalkyl ketones, especially cyclohexanone; cyclic ethers, e.g., dioxane and tetrahydrofuran; acetonitrile; pyridine; dimethylformamide; and dimethylsulfoxide.

Preferred acids (group C) are lower-alkanoic acids containing one to four carbon atoms, e.g., formic acid, acetic acid and propionic acid, and substituted carboxylic acids, especially substituted lower-alkanoic acids, e.g., methoxyacetic acid. The separations are generally less successful with mineral acids, which are thus not preferred. The proportion of the acid in the total mixture normally is between 1 and 40 percent by volume, preferably about 5-20 percent by volume.

If the simultaneous separation of the less polar metabolites, e.g., homovanillic acid is not desired, it is also possible to omit one of the above-mentioned group A or group B solvents from the eluent mixture.

According to a particularly preferred embodiment, the eluent employed is a mixture of chloroform/n-butanol/ethanol/ glacial acetic acid/water, especially in a volume ratio of about 5/55/10/15/15. Other suitable mixtures are, for example, n-butanol/isopropanol/glacial acetic acid/water, e.g., in a volume ratio of about 60-40/10-30/15/15, e.g., about 60/10/15/15, and about 40/30/15/15; n-butanol/ethyl acetate/glacial acetic acid/water, e.g., in a volume ratio of about 60/10/ 15/ 15; n-butanol/glacial acetic acid/water; e.g., in a volume ratio of about 70/ 15/ 15; and n-butanol/ethanol/glacial acetic acid/water, e.g., in a volume ratio of about 60/5/ /25, respectively.

The viscosity of the eluent mixture at 20 C. is preferably between 0.2 and 5 centipoises. lts boiling point range is preferably between 30 and 150 C.

In a preferred embodiment of the process of this invention, the separation of the components of the body fluid by the eluents is conducted two to four times. In such a case, it is possible to employ, in the second or in any of the following separations, an alkaline eluent mixture. However, it is important, when the urine is applied without any pretreatment, which is preferred, that the first separation on the PE] cellulose be conducted with an acidic eluent mixture.

The further treatment of the resulting chromatogram, especially in two-dimensional chromatography, can also be conducted with acidic, neutral or basic eluents. Especially satisfactory separations can be obtained, for example, if the basic eluent mixtures employed are those containing the solvent components mentioned above for the acidic eluents, except the acid component is replaced by a basic component, preferably ammonia. The proportion of base in the solvent mixture is normally between 1 and 40 percent by volume, preferably about 5-30 percent by volume.

The basic eluent mixture, in addition to the base, desirably contains about 5 to 20 percent of water; at least one solvent immiscible or miscible to only a slight extent with water (group A); and at least one solvent miscible both with water and with organic solvents (group B). An example of a satisfactory mixture is ethyl acetate/n-butanol/isopropanol/25 percent aqueous ammonium hydroxide, preferably in a volume ratio of about 30/20/25/25. Here again, one of the solvent groups A or B can be omitted, since, for many problems, a sufficient separation is nevertheless effected. Examples for further basic eluent mixtures are the following: Chloroform/nbutanol/ethanol/25 percent aqueous ammonium hydroxide/water, preferably in a volume ratio of about 20/30/30/ 10; ethyl acetate/n-butanol/isoproanol/25 percent ammonium hydroxide, preferably in a volume ratio of about 30/20/25/25;

n-butanol/acetone/25 percent ammonium hydroxide/water, preferably in a volume ratio of 70/ 10/10/ 10; n-butanollmethyl ethyl ketone/25 percent hydroxide/water, preferably in a volume ratio of about 30/40/15/15; isoamyl acetate/isopropanolZS percent ammonium hydroxide, preferably in a volume ratio of 30/45/25; and isopropanol/25 percent ammonium hydroxide, preferably in a volume ratio of /20.

The production of the thin-layer chromatographic plates to be employed for conducting the process of this invention is effected in a conventional manner. See, e.g., Bobbitt, James M., Thin-Layer Chromatography (1963) Reinhold Publishing Corp., N. Y. The adsorbent suspension is applied by means of a conventional spreader to the substrates of glass, synthetic films, metallic foils, or other suitable materials. The thickness of the adsorbent layer normally ranges between 50 and 200 u. The plates are then dried for several hours at room temperature, or for a shorter period of time at an elevated temperature, e.g., several minutes at 80-l30 C. Since the layer is light-sensitive, it is desirable to store the plates so that they are protected from light.

The body fluid to be assayed for catecholamine and serotonin metabolites usually is urine. Basically, however, it is also possible to conduct the determinations with other suitable body fluids, e.g., cerebrospinal fluid.

The novel process is conducted in a conventional manner. The urine or other body fluid is applied to the adsorbent layer, preferably without any pretreatment. In case of a quantitative determination, the urine specimen is, in certain cases, diluted, e.g., with deionized water, in order to employ comparable volumes, and to facilitate the mathematical conversion. Suitably, for example, the excretion for a 24 hour period is, in each case, diluted to the nearest full liter, e.g., 1,200 to 1,600 cc of urine would be diluted to 2 liters.

Approximately l-S pl. of each urine sample are applied to the adsorbent layer. It is advantageous to conduct an intermediate drying step if more than 2 #1. are applied to one point. It is desirable to apply one standard solution containing known concentrations of the components being assayed for in the specimens alongside each 23 urine specimens. For developing the plates, one or several are inserted in customary development chambers containing the element mixture. Suitably, the chamber is closed during the developing step by means of a cover plate. Usually, the eluent front reaches the upper rim of the plate in about l-2 hours (height of ascent 8.5 cm., with aplate size of 10 X10 cm. or 20 X10 cm.).

Most advantageously, a second development is then conducted in the same manner after a drying step, optionally using the same or another acidic eluent or a neutral or basic eluent.

In order to identify the substances contained in the tested fluid and in the standard, an appropriate color reagent is thereafter sprayed on the developed plate in the usual manner. Normally, these color reagents are employed in concentrations of about 0.1-1 percent, usually in stabilized form. Most frequently, diazotized p-nitroanilines are utilized, e.g., pnitrophenyl-diazonium fluoroborate, 4-diazobenzenesulfonic acid, 4diazo-N-monoethyl-orthotoluidine fluoroborate, 4- diazo-N,N-diethylaniline fluoroborate or 4-diazo-N-ethyl-N- (B-hydroxyethyl)-aniline. 2,6-dichloroquinone chlorimide has also been employed for this purpose. During spraying, care should be taken that the adsorbent layer is just barely moistened, since otherwise the zones can run into one another. After drying, e.g., for a few minutes at C., or correspondingly longer at lower temperatures, the chromatogram is evaluated in direct illumination and trans-illumination. The height and the color of the spots of the urine chromatograms are compared with those of the standard solutions. Vanillic acid, metadrenaline, normetadrenaline and methoxyhydroxymandelic acid exhibit blue colorations and homovanillic acid shows a light-blue coloration when p-nitrophenyl-diazonium fluoroborate is employed as the color reagent. In contrast thereto, hydroxyindoleacetic acid is colored red with this reagent. Pathological amounts of secretion of the catecholamine and serotonin metabolites can be recognized with accuracy in this manner.

In many cases, it is advantageous to further develop the thus-obtained chromatograms, especially in case of a positive finding, by means of two-dimensional chromatography. This increases the specificity of the assay to a considerable extent, since the spots obtained in the one-dimensional chromatography are once again separated. Due to the intake of medicines or also due to alimentary causes, problems can arise in the determination of the metabolites because other metabolites can be present which sometimes have the same R value as catecholamine and serotonin metabolites in the chosen eluting agent. Therefore, it is often advisable to confirm a' positive finding in the thin film chromatography by twodimensional chromatography. A definite conclusion can then be drawn whether the apparent increased concentration of a metabolite was merely simulated by the presence of another substance with the same R value in the body fluid being examined. The two-dimensional chromatography is preferably conducted twice in each dimension. Here, also, it is optionally possible to employ a basic eluting agent in the second dimension and/or during the second development, respectively.

Advantageously, the layer is divided into two parts in twodimensional chromatography. First, two spatially separated urine specimens and a standard solution are developed in the usual manner. Then a second standard is applied and chromatography in the second dimension is then conducted in a conventional manner.

The spots developed from the urine specimens obtained in this fashion can now readily be identified by reference to the two corresponding spots of the standard solutions, i.e., by means of two coordinates. Also, the quantitative assessment of the separated substances can be conducted with great certainty in this manner.

By the present invention, it is now possible for the first time to conduct a side-by-side identification and quantitative as sessment of both Catecholamine and serotonin metabolites, without previously having to treat the urine, thereby eliminating a source of error. Because of its simplicity and rapidity of its conductance, the novel process is suitable for series investigations as well as for individual determinations in case of pathological findings. The low detection limits of the process render it useful for a great variety of problems and differential diagnoses.

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

MANUFACTURE OF THE THIN-LAYER PLATES Three liters of desalted water is added to 150 g. of a 50 percent aqueous solution of polyethylenimine. The solution is transferred to a larger vessel and replenished in this vessel with desalted water to a total of 17.5 kg. 4.8 kg. of microcrystalline cellulose is added to this polyethylenimine solution. The mixture is suspended in a mixer at a high speed for 20 seconds. Glass plates of 20 X 20 cm. are coated with the thusobtained suspension. The spreading device is adjusted to a layer thickness of 250 u. The plates are dried in a drying CONDUCT ANCE OF THE PROCESS EXAMPLE 1 Use As A Clinical Test-For The Recognition Of Increased Renal Excretion of Catecholamine and Serotonin Metabolites The collected urine of 24 hours of a patient is brought with distilled water to a volume of l, 2, or 3 liters. Two pl. of urine per 1 liter of the standardized urine sample is applied in dots to the longitudinal side of a PEI cellulose glass plate of a size of 10 X 20 cm. at a distance of 1.5 cm. from the lower edge. Between the individual applied dots, a spacing of 1.5 cm. is maintained. Beside each second urine specimen, a standard solution (see table below for the composition thereof) is applied. In this manner, each urine sample is adjacent a standard solution, and eight urine specimens can be applied to a plate. The plate is now developed twice up to the upper edge of the plate with the eluent mixture of chloroform/butanol/ethano l/glacial acetic acid/water (5/55/10/15/15). After the first development, the plate is taken out of the development chamber, is dried in a stream of warm air, and than is placed again into the development chamber.

The plate is then dried and sprayed with a ODS-molar solution of p-nitrobenzene-diazonium tetratluoroborate in a 0.3- molar aqueous Na cO solution. After a short period of heating, the individual metabolites appear separately as spots of various colors. On the basis of the intensity of the colorations and the size of the spots, the determination is made whether one of the urine specimens contains a metabolite at a higher concentration than the standard.

The standard is produced by dissolving known amounts of the respective metabolites in standard volume of water. The concentrations of the metabolites in the standard correspond to the upper normal range, except for metadrenaline and normetadrenaline, which were added in excess amounts to improve recognition. The composition of the standards, as well as the ascent levels of the individual standard substances in the eluent employed is set forth in Table l.

All of the selected urine specimens which were examined against this standard exhibited values lying within the normal sens? EXAMPLE 2 Semiquantitative Assessment of the Renal Excretion of Catecholamine and Serotonin Metabolites Standard solutions are prepared having the following composition and concentration:

TABLE 2 [Concentration of the standard solution (ST) in mg./1]

S'11 S'I2 S'13 8T4 8T5 In an alternate fashion, 2 pl. of urine per one liter of daily excretion and one of the above standard solutions are applied to the chromatographic plate, the latter in the increasing concentration, in the manner described in Example 1. The plate is developed and the color reaction is conducted as described in Example 1. With reference to the standards of varying concentration, the amount of the various metabolites secreted can be assessed without difficulty and with a great amount of accuracy.

Four of the urine specimens which were compared against these standards exhibited values in the normal range for the six above-mentioned metabolites. One specimen, which contained an excessive amount of homovanillic acid, had an increased concentration which was classified between Standard 2 and Standard 3 (light-blue spot). This corresponded to a concentration of 25 mg./l. of homovanillic acid in that urine specimen.

EXAMPLE 3 Two-Dimensional Chromatography A X 20 cm. glass plate coated as described above with PEI cellulose is divided by a pencil line into two 10 X 10 cm. halves. To the thus-obtained left-hand portion of the plate, at least 4 pl. of an untreated urine is applied at a distance of 1.5 cm. from the left-hand and lower edges, respectively. On the right-hand half of the plate, the application is carried out correspondingly, but in this case 2 pl. of a standard solution (ST-3; Example 2) is also applied at a spacing of mm. from the point of application of the urine sample. The plate is developed twice in the usual manner with intermediate drying by ascending chromatography to the upper edge of the plate with the longitudinal side of origin downwardly using as eluent chloroform/butanol/ethanol/ glacial acetic acid/water (5/55/10/15/15).

After another intermediate drying step, 2 pl. of the standard solution ST-3 is applied at the point where the urine was applied in the left-hand portion of the plate or closely adjacent thereto. Then, the left-hand half of the plate is developed twice, with intermediate drying, at right angles to the first developing direction up to the upper end, i.e., up to the pencil line, with an eluent mixture consisting of ethyl acetate/n-butanol/isopropanol/25 percent aqueous ammonium hydroxide (30/20/25/25).

The thus-obtained spots are made visible by spraying with a 0.05-molar solution of p-nitrophenyl-diazonium fluoroborate. Each spot can be unequivocally characterized in two coordinates by the standard solutions employed in the two-dimensional development procedure.

In this particular sample, it became clear from the twodimensional chromatogram that the apparently abnormally high concentration of hydroxymethoxymandelic acid (blue spot), detected in the preceding one-dimensional chromatogram was actually simulated by the presence of another metabolite having the same ascent level. The spot of hydroxymethoxymandelic acid obtained in the two-dimensional chromatogram was in the normal range with respect to color and size.

EXAMPLE 4 In the manner described in Example 1, I00 24-hour urine specimens were investigated. In a variation from Example 1, the standard solution was replaced by a standard solution containing I-IMMS, HIES, I-IVS, and VS, each in concentrations of 5 mg./l.

After the development and coloring of the chromatograms in the manner described in Example 1, the following results were obtained:

Of the 100 urine specimens, 22 urine specimens contained approximately 5 mg./l. of HIES, 2 urine specimens contained about 7 mg./l. of HIES, and the remainder of the urine specimens contained less than 5 mg./l. of I-IIES. All urine specimens containing 5 mg./l. of I-IIES or more were subjected to two-dimensional chromatography in the manner described parent I-IIES level above 5 mg./l. observed in the one-dimensional chromatogram a simulated elevated level.

Twenty-one of the urine specimens which were examined contained, as evaluated by one-dimensional chromatogram, 5 mg./l. or more of l-IMMS. A re-examination by two-dimensional chromatography of these specimens revealed that an interfering substance was present in 5 specimens, which interfering substance was separated by two-dimensional chromatography. From this, it was determined that the HMMS excretion in these five samples was within normal range. Accordingly, only 16 of the urine specimens examined actually exhibited a slightly increased excretion of I-IMMS, which fact could be confirmed by other tests.

HVS was discovered only in 10 of the 100 urine specimens by the size and intensity of the light-blue spots. In may urine specimens, in one-dimensional chromatography, a substance having a red coloration was found just beneath the I'IVS spots. In order to be absolutely sure that this red color substance did not cover a spot caused by HVS, 5, l0, l5 and 20 mg, respectively, of I-IVS per liter were added to the separate portions of a urine specimen containing this substance. It was found after the development of the chromatogram that the detection of HVS in the process of this invention is not impaired by this substance which assumes a red coloration, because the differences in HVS concentration could be clearly seen. In one case, a urine sample contained a substance assuming a red color at exactly the development point of l-IVS. However, twodimensional chromatogram of the specimen showed that the specimen did not contain any substantial amounts of HVS.

Accordingly, the process of this invention did not erroneously indicate a pathological excretion in any of the 100 urine specimens examined, which established that erroneously positive results are not likely. This is even more surprising in view of the fact the urine samples were obtained from patients who were not readied for the test by control of diet or ingestion of drugs. Erroneous negative results are likewise unlikely, due to the high sensitivity of metabolites detection according to the process of this invention.

EXAMPLE 5 Semiquantitative Assessment of the Renal Excretion of Catecholamine and Serotonin Metabolites Standard solution having the composition and concentration set forth in Example 2 are prepared.

From a urine sample found to have a striking increased content of homovanillic acid, there are applied alternately 2 pl. of urine per one liter of daily excretion and respectively one of the various standard solutions in increasing concentrations, in the manner described in Example 1. The plate is developed and the color reaction carried out as described in Example 1. By reference to the standards of varying concentration, the amounts of the various metabolites in the urine sample can be assessed without difiiculty and with great accuracy.

By comparison of the intensities of the visualized homovanillic acid spots, the urine specimen was classified between Standard 2 and Standard 3 (light-blue spot), which corresponds to a homovanillic acid content of 25 mg./l. of the unne specimen.

The preceding examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions of this invention for those used in the preceding examples.

From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

What is claimed is:

1. A process for the determination of catecholamine and serotonin metabolites in body fluids by thin-layer chromatog- 9 raphy which comprises using as the chromatographic adsorbent a cellulose impregnated with polyethylenimine.

2. A process according to claim 1 wherein the cellulose is substantially free of anions having a selectivity coefficient as high as the chloride ion.

3. A process according to claim 1 wherein the adsorbent is microcrystalline cellulose impregnated with polyethylenimine.

4. A process according to claim 1 wherein the chromatogram is developed using as eluent a solvent mixture comprising at least three solvents.

5. A process according to claim 1 wherein the chromatogram is developed from two to four times.

6. A process according to claim 1 wherein at least the first development of the chromatogram is conducted with an acidic eluent mixture.

7. A process according to claim 1 wherein the chromatogram is developed with an eluent mixture which contains at least 5 percent of water and at least one solvent from each of the following groups:

a. solvents which are substantially immiscible with water;

b. solvents miscible both with water and with organic solvents; and

c. acids.

8. A process according to claim 7 wherein the acid is a lower-alkanoic acid.

9. A process according to claim 7 wherein the eluent solvent is a mixture of chloroform, n-butanol, ethanol, glacial acetic acid and water.

10. A process according to claim 9 wherein the solvents are employed in a volume ratio of about 5/55/ 10/ /15, respectively.

11. A process according to claim 7 wherein at least one development of the chromatogram after the first development is conducted with a basic eluent mixture.

12. A process according to claim 11 wherein the chromatogram is developed with an eluent solvent mixture which contains at least 5 percent of water and at least one further solvent from each of the following groups:

a. solvents which are substantially immiscible with water;

b. solvents miscible both with water and with organic solvents;

c. a base.

13. A process according to claim 12 wherein the solvent mixture comprises a volatile base.

14. A process according to claim 13 wherein the solvent mixture comprises ammonia.

15. A process according to claim 12 wherein the eluent mixture is a mixture of ethyl acetate, n-butanol, isopropanol and aqueous ammonium hydroxide.

16. A process according to claim 15 wherein the solvents are employed in a volume ratio of 30/20/25/25, respectively.

17. A process according to claim 1 wherein the chromatography is two dimensional.

18. A process according to claim 1 wherein the chromatography is first conducted in one dimension and then conducted in a second dimension using a basic eluent mixture.

19. A process according to claim 1 wherein the body fluid is untreated urine. 

2. A process according to claim 1 wherein the cellulose is substantially free of anions having a selectivity coefficient as high as the chloride ion.
 3. A process according to claim 1 wherein the adsorbent is microcrystalline cellulose impregnated with polyethylenimine.
 4. A process according to claim 1 wherein the chromatogram is developed using as eluent a solvent mixture comprising at least three solvents.
 5. A process according to claim 1 wherein the chromatogram is developed from two to four times.
 6. A process according to claim 1 wherein at least the first development of the chromatogram is conducted with an acidic eluent mixture.
 7. A process according to claim 1 wherein the chromatogram is developed with an eluent mixture which contains at least 5 percent of water and at least one solvent from each of the following groups: a. solvents which are substantially immiscible with water; b. solvents miscible both with water and with organic solvents; and c. acids.
 8. A process according to claim 7 wherein the acid is a lower-alkanoic acid.
 9. A process according to claim 7 wherein the eluent solvent is a mixture of chloroform, n-butanol, ethanol, glacial acetic acid and water.
 10. A process according to claim 9 wherein the solvents are employed in a volume ratio of about 5/55/10/15/15, respectively.
 11. A process according to claim 7 wherein at least one development of the chromatogram after the first development is conducted with a basic eluent mixture.
 12. A process according to claim 11 wherein the chromatogram is developed with an eluent solvent mixture which contains at least 5 percent of water and at least one further solvent from each of the following groups: a. solvents which are substantially immiscible with water; b. solvents miscible both with water and with organic solvents; c. a base.
 13. A process according to claim 12 wherein the solvent mixture comprises a volatile base.
 14. A process according to claim 13 wherein the solvent mixture comprises ammonia.
 15. A process according to claim 12 wherein the eluent mixture is a mixture of ethyl acetate, n-butanol, isopropanol and aqueous ammonium hydroxide.
 16. A process according to claim 15 wherein the solvents are employed in a volume ratio of 30/20/25/25, respectively.
 17. A process according to claim 1 wherein the chromatography is two dimensional.
 18. A process according to claim 1 wherein the chromatography is first conducted in one dimension and then conducted in a second dimension using a basic eluent mixture.
 19. A process according to claim 1 wherein the body fluid is untreated urine. 